Anti-coronavirus compounds

ABSTRACT

A method of treating coronavirus infection. The method includes administering to a subject suffering from or being at risk of suffering from such infection an effective amount of a compound of formula (I). Each variable in this formula is defined in the specification.

CROSS REFERENCE TO RELATED APPLICATION

Under 35 U.S.C. §119, this application claims priority to U.S.Provisional Application Ser. No. 60/568,080, filed May 4, 2004, thecontents of which are incorporated herein by reference.

BACKGROUND

Coronavirus is the cause of many common colds in humans. Recently,infection in humans with a new coronavirus led to a worldwide outbreakof an acute respiratory disease, i.e., severe acute respiratorysyndrome. See, e.g., Ksiazek et al., N. Engl. J. Med., 2004, 348:1953-1966. The first severe acute respiratory syndrome case wasidentified in Guangdong, China in November, 2002. The disease laterspread to more than 25 countries. By Jul. 31, 2003, about 8,100 severeacute respiratory syndrome cases and about 800 severe acute respiratorysyndrome-related deaths were reported around the world.

Various drugs have been investigated for use in treating severe acuterespiratory syndrome. They include ribavirin, corticosteroids, Kaletra,glycyrrhizin, and certain human interferons. See, e.g., Peris et al.,Lancet, 2003, 361:1319; Cinatl et al., Lancet, 2003, 361:2045; andCinatl et al., Lancet, 2003, 362:293. However, these drugs require highdosages to exert efficacy. Thus, there exists a need to identifycompounds that can more effectively treat severe acute respiratorysyndrome.

SUMMARY

This invention is based on an unexpected discovery that certaincompounds are effective in treating an infection with a coronavirus(e.g., a severe acute respiratory syndrome virus).

In one aspect, this inventions features a method for treating a viralinfection. The method includes administering to a subject (e.g., amammal) suffering from or being at risk of suffering from an infectionwith a coronavirus (e.g., a severe acute respiratory syndrome virus) aneffective amount of a compound of formula (I) or a salt thereof:

In this formula, R₁ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, or OR_(a); each of R₂, R₃, R₄ andR₁₀, independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, or aryl; each of R₅ and R₁₁,independently, is alkyl substituted with aryl; each of R₆, R₇, R₈, andR₉, independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, or OR_(b); and R₁₂ is C₁-C₁₀ alkylsubstituted with OR_(c), NHC(O)R_(c), or NHC(O)OR_(c); in which each ofR_(a), R_(b), and R_(c), independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl, or aryl.

Note that one can administer a compound of formula (I) to a subjectsuffering from or being at risk of suffering from an infection with botha coronavirus (e.g., a severe acute respiratory syndrome virus) and ahuman or feline immunodeficiency virus. See, e.g., U.S. Pat. No.6,803,466 and Lee et al., J. Am. Chem. Soc., 1999, 121:1145-1155.

Referring to formula (I), a subset of compounds described above arethose in which R₁ is C₁-C₁₀ alkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl,aryl, or OR_(a); each of R₂, R₃, R₄ and R₁₀, independently, is H orC₁-C₁₀ alkyl; and each of R₆, R₇, R₈, and R₉, independently, is H orOR_(b). In these compounds, R₁ can be C₁-C₁₀ alkyl substituted withC₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl, aryl, OR, SR, orNHC(O)OR; R being H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, or aryl; R₂ can be isopropyl; R₅ and R₁₁,can be alkyl substituted with phenyl; and each of R₆, R₇, R₈, and R₉,independently, can be H or OH.

The term “alkyl” refers to a saturated or unsaturated, linear orbranched hydrocarbon moiety, such as —CH₃, —CH₂—CH═CH₂, or branched—C₃H₇. The term “cycloalkyl” refers to a saturated or unsaturated,non-aromatic cyclic hydrocarbon moiety, such as cyclohexyl orcyclohexen-3-yl. The term “heterocycloalkyl” refers to a saturated orunsaturated, non-aromatic cyclic moiety having at least one ringheteroatom (e.g., N, O, or S), such as 4-tetrahydropyranyl or 4-pyranyl.The term “aryl” refers to a hydrocarbon moiety having one or morearomatic rings. Examples of aryl moieties include phenyl (Ph),phenylene, naphthyl, naphthylene, pyrenyl, anthryl, and phenanthryl. Theterm “heteroaryl” refers to a moiety having one or more aromatic ringsthat contain at least one heteroatom (e.g., N, O, or S). Examples ofheteroaryl moieties include furyl, furylene, fluorenyl, pyrrolyl,thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl,quinazolinyl, quinolyl, isoquinolyl, and indolyl.

Alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl mentionedherein include both substituted and unsubstituted moieties, unlessspecified otherwise. Possible substituents on cycloalkyl,heterocycloalkyl, aryl, and heteroaryl include, but are not limited to,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₈ cycloalkyl, C₅-C₈cycloalkenyl, C₁-C₁₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,amino, C₁-C₁₀ alkylamino, C₁-C₂₀ dialkylamino, arylamino, diarylamino,hydroxyl, halogen, thio, C₁-C₁₀ alkylthio, arylthio, C₁-C₁₀alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl,amidino, guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy,carboxyl, and carboxylic ester. Possible substituents on alkyl includeall of the above-recited substituents except C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, and C₂-C₁₀ alkynyl. Cycloalkyl, heterocycloalkyl, aryl, andheteroaryl can also be fused with each other.

The term “treating” or “treatment” mentioned herein refers toadministering one or more of the drug compounds described herein to asubject, who has an infection with a coronavirus, and possibly, also ahuman or feline immunodeficiency virus, a symptom of such an infection,or a predisposition toward such an infection, with the purpose to confera therapeutic effect, e.g., to relieve, alter, affect, or ameliorate theviral infection, the symptom of it, or the predisposition toward it.Such a subject can be identified by a health care professional based onresults from any suitable diagnostic method. “An effective amount”refers to the amount of the compound(s) required to confer a therapeuticeffect on a treated subject or confer an inhibitory effect on a viralprotease.

In another aspect, this invention features a packaged product includinga container, a compound of formula (I), and a legend associated with thecontainer and indicating administration of the compound for treating aninfection with a coronavirus, such as a severe acute respiratorysyndrome virus. The legend can also indicate administration of thecompound for treating an infection with a human or felineimmunodeficiency virus.

In still another aspect, this invention features a method for inhibitinga viral protease. The method includes contacting a coronavirus protease(such as a severe acute respiratory syndrome virus protease) with aneffective amount of a compound of formula (I). The method can furtherincludes contacting the compound with a human or feline immunodeficiencyvirus protease. See, e.g., U.S. Pat. No. 6,803,466 and Lee et al., J.Am. Chem. Soc., 1999, 121:1145-1155.

In still another aspect, this invention features the compounds offormula (I) or a salt thereof. Referring to this formula, the samegroups as those described above are assigned to each variable exceptthat R₁ is H, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl,aryl, OR_(a), or C₁-C₁₀ alkyl substituted with C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, OR_(a), or SR_(a). A subset of thejust-described compounds are those in which R₁ is C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, OR_(a), or C₁-C₁₀ alkyl substitutedwith C₃-C₂₀ heterocycloalkyl, aryl, OR_(a), or SR_(a); each of R₂, R₃,R₄ and R₁₀, independently, is H or C₁-C₁₀ alkyl; and each of R₆, R₇, R₈,and R₉, independently, is H or OR_(b).

In still another aspect, this invention features a method for treatingan infection with a coronavirus by administering to a subject in needthereof an effective amount of a compound of formula (II) or a saltthereof:

In this formula, R₁ is C₃-C₂₀ cycloalkyl or C₃-C₂₀ heterocycloalkyl;each of R₂, R₃, R₄, R₅, R₆, R₁₁, and R₁₂, independently, is H, C₁-C₁₀alkyl, or OR_(a); each of R₇ and R₁₀ is H; or R₇ and R₁₀, takentogether, are —O—; R₈ is C₁-C₁₀ alkyl or COOR_(b); and R₉ is H, OR_(c),or OC(O)R_(c); in which each of R_(a), R_(b), and R_(c), independently,is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl,heteroaryl, or aryl.

Referring to formula (II), a subset of the compounds described above arethose in which R₁ is tetrahydropyranyl substituted with C₁-C₁₀ alkyl,C₃-C₂₀ heterocycloalkyl, OR, or COOR; R being H, C₁-C₁₀ alkyl, C₃-C₂₀cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl, or aryl; each of R₂,R₃, R₄, R₅, R₆, R₁₁, and R₁₂, independently, is H, OH, or CH₃ optionallysubstituted with OH; or R₈ is CH₃ or COOH.

In still anther aspect, this invention features a method for treating aninfection with a coronavirus by administering to a subject in needthereof an effective amount of a compound of formula (III) or a saltthereof:

In this formula,

is a single bond or a double bond; X is —O— or —C(R_(a)R_(b))—; and eachof R₁, R₂, R₃, R₄, R₅, and R₆, independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl, aryl, OR_(c), orCOOR_(c); in which each of R_(a), R_(b), and R_(c), independently, is H,C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl,aryl, or OC(O)R; R being H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, or aryl.

Referring to formula (III), a subset of the compounds described aboveare those in which

is a single bond. In these compounds, X can be —C(R_(a)R_(b))—; R₁, R₂,R₃, R₄, R₅, and R₆, independently, can be H, OH, OCH₃, or COOCH₃; andeach of R_(a) and R_(b), independently, can be H or OC(O)R. Anothersubset of the compounds described above are those in which

is a double bond. In these compounds, X can be —O— and each of R₁, R₂,R₃, R₄, R₅, and R₆, independently, can be H or COOCH₃.

In yet another aspect, this invention features a method for treating aninfection with a coronavirus (e.g., a severe acute respiratory syndromevirus) by administering to a subject in need thereof an effective amountof compound 3, 4, 6-12, 14, 15, or 17 (structures shown below) or a saltthereof.

In addition, this invention encompasses a pharmaceutical compositionthat contains an effective amount of at least one of the above-describedcompounds and a pharmaceutically acceptable carrier.

The compounds that can be used to practice this invention include thecompounds themselves, as well as their salts, prodrugs, and solvates, ifapplicable. A salt, for example, can be formed between an anion and apositively charged group (e.g., amino) on an above-described compound.Suitable anions include chloride, bromide, iodide, sulfate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate,tosylate, tartrate, fumurate, glutamate, glucuronate, lactate,glutarate, and maleate. Likewise, a salt can also be formed between acation and a negatively charged group (e.g., carboxylate) on anabove-described compound. Suitable cations include sodium ion, potassiumion, magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The above-described compounds also includethose salts containing quaternary nitrogen atoms. Examples of prodrugsinclude esters and other pharmaceutically acceptable derivatives, which,upon administration to a subject, are capable of providing activecompounds described above. A solvate refers to a complex formed betweenan active compound described above and a pharmaceutically acceptablesolvent. Examples of pharmaceutically acceptable solvents include water,ethanol, isopropanol, ethyl acetate, acetic acid, and ethanolamine.

Also within the scope of this invention is a composition containing oneor more of the above-described compounds for use in treating acoronavirus infection, and the use of such a composition for themanufacture of a medicament for the just-mentioned treatment.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

This invention relates to treating an infection with a coronavirus, suchas a severe acute respiratory syndrome virus. Shown below are 35exemplary compounds (i.e., compounds 1-35) that can be used to practicethis invention:

Some of the compounds described above are available from commercialsources, such as Sigma-Aldrich, St. Louis, Mo. All of them can beprepared by known methods. See, e.g., Lee et al., J. Am. Chem. Soc.,1999, 121:1145-1155 and the references cited therein. For instance,compound 25 shown above can be prepared via a series of peptide couplingreactions, which are well known in the art. One of such reactions isdescribed in Example 3 below. Details of preparation of compounds 1, 2,and 26-35 are provided in Examples 1, 2, and 4-13 below. Compounds 3-24are commercially available. A compound thus synthesized can be purifiedby a method such as column chromatography, high-pressure liquidchromatography, or recrystallization. Other similar compounds used topractice this invention can be prepared using other suitable startingmaterials through the above synthetic routes and others known in theart. The methods described above may also include additional steps,either before or after the steps described specifically herein, to addor remove suitable protecting groups in order to ultimately allowsynthesis of the compounds described above. In addition, varioussynthetic steps may be performed in an alternate sequence to give thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingapplicable compounds are known in the art and include, for example,those described in R. Larock, Comprehensive Organic Transformations, VCHPublishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 2^(nd) Ed., John Wiley and Sons (1991); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995) and subsequenteditions thereof.

The compounds mentioned herein may contain a non-aromatic double bondand one or more asymmetric centers. Thus, they can occur as racematesand racemic mixtures, single enantiomers, individual diastereomers,diastereomeric mixtures, and cis- or trans-isomeric forms. All suchisomeric forms are contemplated.

This invention also covers a method of administering an effective amountof one or more of the drug compounds described above to a subject havinga coronavirus infection, and optionally, also having a human or felineimmunodeficiency virus infection. The drug compounds can be screened fortheir inhibitory activities against these infections by methodsdescribed below or by methods known in the art, such as those describedin U.S. Pat. No. 6,803,466 and Lee et al., J. Am. Chem. Soc., 1999,121:1145-1155.

Further, this invention covers a method of contacting an effectiveamount of one or more drug compounds described above with a coronavirusprotease and optionally, with a human or feline immunodeficiency virusprotease or both. The compounds can be screened for their inhibitoryactivities against one or more of these proteases by methods describedbelow or by methods known in the art, such as those described in U.S.Pat. No. 6,803,466 and Lee et al., J. Am. Chem. Soc., 1999,121:1145-1155.

Also within the scope of this invention is a pharmaceutical compositioncontaining an effective amount of at least one compound described aboveand a pharmaceutical acceptable carrier. Regimens for administering sucha pharmaceutical composition are well known and, if necessary, can beeasily re-established. Effective doses will vary, as recognized by thoseskilled in the art, depending on the type or degree of the coronaviralinfection; the subject's size, weight, age, and sex; the route ofadministration; the excipient usage; and the possible co-usage withother therapeutic treatment.

To practice the method of the present invention, a composition havingone or more of the above-mentioned compounds can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional, or intracranialinjection, as well as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordiglycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions, and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A composition havingone or more active above-described compounds can also be administered inthe form of suppositories for rectal administration.

A pharmaceutically acceptable carrier is routinely used with one or moreactive above-mentioned compounds. The carrier in the pharmaceuticalcomposition must be “acceptable” in the sense that it is compatible withthe active ingredient of the composition (and preferably, capable ofstabilizing the active ingredient) and not deleterious to the subject tobe treated. One or more solubilizing agents can be utilized aspharmaceutical excipients for delivery of an above-mentioned compound.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

The compounds described above can be preliminarily screened by in vitroassays for their efficacy against the replication of severe acuterespiratory syndrome virus (See Examples 14 and 15 below). Other methodswill also be apparent to those of ordinary skill in the art. See, e.g.,U.S. Pat. No. 6,803,466 and Lee et al., J. Am. Chem. Soc., 1999,121:1145-1155. These compounds can be further screened by in vivoassays.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

EXAMPLE 1 Preparation of Compound 1

Compound I was prepared according to the method described in Lee et al.,Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 939-944.

Compound I (0.6 g, 1.11 mmol) was dissolved in a solution oftriethylamine and methanol (10% TEA in MeOH, 5.5 mL). A solution ofdi-tert-butyl dicarbonate (0.24 g, 1.11 mmol) in methanol (5 mL) wasthen added dropwise to the above mixture with vigorous stirring. Themixture was stirred at room temperature overnight. The methanol and TEAwere removed in vacuo to yield an oily residue. The residue wasdissolved in dichloromethane (30 mL) and washed with a solution of a 10%sodium carbonate aqueous solution (2×50 mL). The organic layer wasseparated, dried over anhydrous Na₂SO₄, filtered, and concentrated invacuo. The oily residue thus obtained was purified by flash columnchromatography to give the compound II (0.55 g, 78%) as a white solid.

Compound II (0.5 g, 0.78 mmol) was dissolved in dry dichloromethane (10mL) in the presence of diisopropylethylamine (0.2 mL, 1.56 mmol). Aftercooling the solution to 0° C., benzyl chloroformate (0.16 g, 0.94 mmol)was added dropwise slowly. After 20 minutes, the reaction was complete(monitored by TLC). The solution was then added to a saturated sodiumhydrogen carbonate solution (20 mL) and the mixture was extracted bydichloromethane (3×20 mL). The organic layers were combined, dried withanhydrous MgSO₄, and concentrated to afford a crude oil. The crude oilwas used for deprotective reaction without purification. Catalyticamounts of p-TsOH were added to a solution of the above crude oil inMeOH (5 mL). The reaction mixture was heated at 60° C. for 24 hours thendiluted with EtOAc (20 mL). The organic solution was washed with asaturated NaHCO₃ aqueous solution (5 mL), a saturated NaCl aqueoussolution (5 mL), dried over anhydrous MgSO₄, filtered, and concentratedin vacuo. The oily residue thus obtained was then purified by flashcolumn chromatography to give the compound 1 as a white solid (0.6 g,75%).

¹H NMR (400 MHz, DMSO-d6): 0.71 (6H, m), 0.73 (6H, m), 1.5 (9Hs), 2.17(1H, se, J=6.7), 2.8-2.9 (3H, m), 3.34 (1H, m), 4.03 (2H, dd, J=8.8,6.4), 4.10 (1H, qu, J=7.0), 4.27 (1H, s), 4.0-4.20 (1H, m), 4.23-4.32(1H, m), 6.92-6.96 (1H, br), 7.05-7.34 (12H, m).

¹³CNMR (100 MHz, DMSO-d6, 80° C.), 17.7, 19.6, 19.7, 28.7, 30.4, 30.5,30.7, 38.3, 45.4, 47.8, 53.0, 60.9, 65.9, 67.1, 72.8, 126.9, 127.6,128.0, 128.8, 128.9, 129.0, 129.7, 138.3, 172.0, 172.5.

MS (ESI) (M+H⁺): 733.

EXAMPLE 2 Preparation of Compound 2

Compound 2 was prepared according to the method described in Lee et al.,Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 939-944.

¹H NMR (400 MHz, DMSO-d6, 80° C.): 0.70 (3H, d, J=2.4), 0.72 (3H, d,J=2.4), 1.21 (3H, d, J=7.0), 1.87 (1H, se, J=6.7), 2.69-2.79 (2H, m),3.32 (1H, s), 4.03 (1H, dd, J=8.8, 6.4), 4.10 (1H, qu, J=7.0), 4.27 (1H,s), 4.34-4.40 (1H, m), 5.04 (2H, s), 6.92-6.96 (1H, br), 7.05-7.34 (12H,m).

¹³C NMR (100 MHz, DMSO-d6, 80° C.): 17.3, 17.6, 18.7, 29.7, 38.0, 49.9,50.4, 57.8, 65.1, 72.5, 125.1, 127.0, 127.2, 127.3, 127.8, 128.6, 136.6,138.4, 155.1, 169.8, 171.6.

HRMS (FAB+) (M+Cs⁺): 1041.3780.

EXAMPLE 3 Preparation of Compound 25

Compounds I and II were respectively prepared according to the methodsdescribed in Stuk et al., J. Org. Chem. 1994, 59:4040 and Sham et al.,Bioorg. Med. Chem. Lett. 2002, 12:1185-1187. To a solution of compound I(0.93 g, 2.0 mmol) and compound II (0.38 g, 2.1 mmol) in 20 ml dry DMFwas added HBTU (a peptide coupling reagent, 0.80 g, 2.1 mmol) followedby DIEA (0.5 mL, 4.2 mmol) at 20° C. under Ar atmosphere. After stirringfor 30 minutes, the reaction mixture was quenched by addition of brineand then extracted with EtOAc. After washing the organic layer with 1 MHCl, a saturated NaHCO₃ aqueous solution, and brine, it was dried overanhydrous MgSO₄, filtered, and concentrated in vacuo. The crude productthus obtained was purified by flash chromatography to give the compoundIII in a 90% yield.

Compound III (0.6 g, 0.98 mmol) in EtOAc (40 mL) was stirred under H₂ (1atm) in the presence of 10% Pd/C (200 mg) at 20° C. for 20 hours. Themixture was filtered through celite and then concentrated in vacuo togive compound IV (0.39 g) as a colorless viscous oil, which was used forcoupling reaction without purification.

HBTU (0.36 g, 0.96 mmol) and DIEA (0.23 mL, 1.91 mmol) were added to asolution of compound IV (0.39 g, 0.87 mmol) and N-Cbz-L-leucine (0.25 g,0.96 mmol) in DMF (15 mL). The reaction mixture was stirred for 30minutes at 20° C. under Ar. After quenching the reaction by addition ofbrine (45 mL), the mixture was extracted with EtOAc (4×30 mL). Theorganic layers were combined and washed sequentially with 1 M HCl (10mL), a saturated NaHCO₃ aqueous solution (10 mL), and a saturated NaClaqueous solution (10 mL). The organic phase was then dried overanhydrous MgSO₄, filtered, and concentrated in vacuo. The crude productthus obtained was purified by flash chromatography to give compound V(0.53 g, 78% two steps) as a white solid.

Compound V (0.5 g, 0.728 mmol) in EtOAc (40 mL) was stirred under H₂ (1atm) in the presence of 10% Pd/C (200 mg) at 20° C. for 20 hours. Themixture was filtered through celite and then concentrated in vacuo. Thecrude product thus obtained was purified by flash chromatography to givecompound VI (0.37 g, 92%) as a white solid.

HBTU (0.15 g, 0.40 mmol) and DIEA (0.1 mL, 0.80 mmol) were added to asolution of compound VI (0.2 g, 0.36 mmol) and5-nitro-furan-2-carboxylic acid (62.8 mg, 0.40 mmol) in DMF (15 mL).After stirring for 30 minutes at 20° C. under Ar, the reaction wasquenched by addition of brine (45 mL) and the mixture was extracted withEtOAc (4×30 mL). The organic layers were combined and washedsequentially with 1 M HCl (10 mL), a saturated NaHCO₃ aqueous solution(10 mL), and a saturated NaCl aqueous solution (10 mL). The organicphase was then dried over anhydrous MgSO₄, filtered, and concentrated invacuo. The crude product thus obtained was purified by flashchromatography to give compound 25 (0.23 g, 90%) as a white solid.

LC-MS (M+H⁺): 699.

EXAMPLE 4 Preparation of Compound 26

Compound 26 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 747.

EXAMPLE 5 Preparation of Compound 27

Compound 27 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 678.

EXAMPLE 6 Preparation of Compound 28

Compound 28 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 711.

EXAMPLE 7 Preparation of Compound 29

Compound 29 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 703.

EXAMPLE 8 Preparation of Compound 30

Compound 30 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 733.

EXAMPLE 9 Preparation of Compound 31

Compound 31 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 732.

EXAMPLE 10 Preparation of Compound 32

Compound 32 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 720.

EXAMPLE 11 Preparation of Compound 33

Compound 33 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 731.

EXAMPLE 12 Preparation of Compound 34

Compound 34 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 752.

EXAMPLE 13 Preparation of Compound 35

Compound 35 was prepared in a manner similar to that described inExample 3.

LC-MS (M+H⁺): 744.

EXAMPLE 14 Screening Assays

Compounds 1-35 were initially screened for their inhibitory activityagainst severe acute respiratory syndrome virus by observingcytopathogenic effect (CPE). The assay procedures are as follows: VeroE6 cells (2×104/well) were cultured in a 96-well plate in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum(FBS). The culture medium was removed after one-day incubation when thecells reached 80-90% confluence. Solutions of 100 μL of DMEM with 2% FBScontaining 10 μL of a test compound were placed in wells (intriplicate). Cells in DMEM with 2% FBS were used as a CPE-positivecontrol (also in triplicate). All of the cells were then incubated in aCO₂ incubator at 37° C. for 2 hours, followed by inoculation with severeacute respiratory syndrome virus (H.K.) at a dose of 100 TCID₅₀/well.The cytopathic morphology of the cells was examined using an invertedmicroscope 72 hours after the infection.

The inhibitory activity of the test compounds was confirmed by usingimmunofluorescence enzyme-linked immunosorbent assay (ELISA),immunofluorescence assay (IFA), western blot analysis (WBA), flowcytometry analysis (FCA), and 3CL-protease inhibition assay. A detaileddescription of these assays are provided below:

Immunofluorescence Enzyme-Linked Immunosorbent Assay

After incubating Vero E6 cells with severe acute respiratory syndromevirus and a test compound, cells were rinsed with phosphate-bufferedsaline (PBS), fixed in a solution containing ice methanol:acetone=1:1for 3 minutes at room temperature, and rinsed three times with PBS. Thecells were then blocked with 3% skimmed milk in PBS for 30 minutes atroom temperature and incubated for 1 hour at 37° C. with 1:2,000 dilutedmonoclonal antibody (ascetic fluid) to the spike protein of severe acuterespiratory syndrome virus. All samples were washed with PBS-T bufferthree times and PBS buffer twice at room temperature, followed by a30-minute incubation with HRP-labeled goat anti-mouse IgG for 30 minutesat room temperature. Plates were rinsed with PBS containing 0.05% Tween20 between incubations. A substrate solution containingO-phenylenediamine dihydrochloride, citrate buffer (pH 5.0), andhydrogen peroxide was added to each well. The plates were then coveredand gently shaken at room temperature for 10 minutes. After the reactionwas stopped by addition of 3N sulfuric acid, the fluorescence intensityof each plate was measured immediately at 492 nm. The EC₅₀ value foreach agent was extrapolated from the linear regression plot of agentconcentration versus OD₄₉₂.

Immunofluorescence Assay

Infected or control cells were rinsed with PBS and re-suspended to afinal concentration of 1×10⁶ cells/mL. Slides were prepared for IFA byspotting wells with 2×10⁴ cells for each test compound concentration orfor each control. The slides were then dried, fixed in an icemethanol:acetone=1:1 solution for 3 minutes, rinsed and stored at −20°C. before staining for IFA. Daudi cells were rehydrated and then blockedwith 3% skimmed milk in PBS for 30 minutes at room temperature. Vero E6cells were rehydrated, blocked, and permeabilized in PBS containing 0.1%saponin and 1% FBS. All other cells were rehydrated, blocked, andpermeabilized in PBS containing 5% FBS, 4% normal goat serum and 0.5%DMSO for 30 minutes. The cells were then incubated in a hydrationchamber at 37° C. for 1 hour with a primary antibody diluted in ablocking solution. Following a rinse with PBS, cells were incubated at37° C. for 1 hour with 3 g/mL FITC-conjugated goat anti-mouse IgG+IgMsecondary antibody (Jackson ImmunResearch, West Grove, Pa.). Afterrinsing with PBS again, cells were stained with 0.1% Evans blue dye(Fisher, Fair Lawn, N.J.) in PBS for 5 minutes. Slides were rinsed toremove any excess contrast dye and cover slides were mounted using asolution of 50% glycerol in PBS. The cells were observed under a Nikon(Nikon, Melville, N.Y.) fluorescence microscope at a magnification of400×. For each test compound concentration, 500 cells were counted andthe percentage of antigen-positive cells was calculated. Theconcentration required to inhibit 50% virus replication (EC₅₀) wasdetermined.

Western Blot Analysis

Severe acute respiratory syndrome virus infected Vero E6 cells weretreated with a test compound at various concentrations for 24 or 48hours and then lysed in a lysis buffer for 3 minutes. The cell debriswas spun down and all cell lysates were harvested for electrophoresisand western blotting assay with SDS-PAGE and a Hybond-C Extra membrane(Amersham Biosciences, Piscataway, N.J.). The resulting membrane wasblocked in 3% skimmed milk in PBS for 30 minutes at room temperature,and then treated with either 1:5,000 diluted anti-spike proteinmonoclonal ascetic fluid or 1:2,000 diluted mouse anti-actin Ab(Chemicon MAb 1501) for 1 hour at room temperature. The membrane wasrinsed using two batches of PBS-T buffer and then washed once for 15minutes and twice for 5 minutes with PBS at room temperature. Themembrane was then treated with 1:2,000 diluted HRP-labeled goatanti-mouse IgG for 30 minutes and for 1 hour. The membrane was washed asabove and a mixed ECL detection reagent was added to the protein side ofthe membrane. The blot was placed in a film cassette with the proteinside up to observe the level of protein expression.

Flow Cytometry Analysis

Vero E6 cells were rinsed and blocked with 5% FBS and 4% goat serum inPBS. Severe acute respiratory syndrome virus infected cells weretrypsinized with 0.05% trypsin-EDTA medium. About 1-5×10⁵ cells weredistributed to each well of a round-bottom ELISA plate or to each 1.5 mLmicrotube containing cell culture medium. The suspension was centrifugedand the cells were rinsed with PBS and resuspended. 100 μL ofCYTOFIX/CYTOPERM solution was added to each well or microtube. The cellswere fixed and permeabilized in 2 mL methanol for 20 minutes at 4° C.The cells were then blocked in PBS containing 5% FBS, 4% serum, and 0.5%DMSO for 30 minutes at 37° C. The cells were incubated with 20 mL ofprimary antibody diluted in a blocking solution for 1 hour at 37° C.,then rinsed twice with 4 mL of the blocking solution and pelleted bycentrifugation at 1000×g for 5 minutes. After the second rinse, 0.5 mLof 3 g/mL FITC conjugated goat anti-mouse IgG+IgM (JacksonImmunoResearch, West Grove, Pa.) was added, followed by incubating thecells for 1 hour at 37° C. The cells were then rinsed twice with PBS andpelleted by centrifugation before being resuspended.

Flow cytometry data was acquired using a Becton-Dickenson FacsCaliburinstrument and analyzed by a Win-MDI 2.7 data analysis program (ScrippsResearch Institute, La Jolla, Calif.). The resulting dot plots weregated to remove non-specific and background staining and an M1 bar wasset so that less than 1% of the cells in the negative control wereincluded in the determination of the percentage of the positive cells.The EC₅₀ value for each test compound was extrapolated using a plotdrawn based on the compound concentration versus the percentage ofantigen positive cells.

3CL-Protease Inhibition Assay

The gene encoding the main protease of severe acute respiratory syndromevirus was cloned from the viral whole genome by using polymerase chainreaction with the forward primer as 5′-GGTATTGAGGGTCGCA-GTGGTTTTAGG-3′and reverse primer as 5′-AGAGGAGAGTTAGAGCCTTATTGG-AAGGTAACACC-3′. ThePCR products flanked by the two primers were subcloned into thepET32Xa/Lic vector. The FXa cleavage site (IEGR) and the complementarysequences to the sticky ends of the linear vector pET-32Xa/LIC wereincluded in these primers. The recombinant protease plasmid was thenused to transform E. coli JM109 competent cells that were streaked on aLuria-Bertani (LB) agar plate containing 100 μg/mL ampicillin. Thecorrect construct was subsequently transformed into E. coli BL21 forexpression of the His-tagged protein, which was then digested with FXaprotease to remove the tag. The structure of the purified protein wasconfirmed by N-terminal sequencing and mass spectrometry. The enzymeconcentration used in all experiments was determined from the absorbanceat 280 nm.

All kinetic measurements were performed in 20 mM Bis-Tris (pH 7.0) at25° C. Enhanced fluorescence due to cleavage of the fluorogenicsubstrate peptide (Dabcyl-KTSAVLQ-SGFRKME-Edans) was monitored at 538 nmwith excitation at 355 nm using a fluorescence plate reader (FluoroskanAscent from ThermoLabsystems, Sweden).

Compound 1-35 were tested using the above-described assays.Unexpectedly, all of them showed low EC₅₀ values, i.e., between 0.85 μMand 100 μM.

EXAMPLE 15 Cytotoxicity Assay

Vero E6 cells were grown in a humidified 5% CO₂ incubator at 37° C. inDMEM supplemented with L-glutamine, non-essential amino acids, and 10%FBS in 75 cm³ flasks. The cells were then seeded at 7×10⁴ cells ml⁻¹onto a 96-well plate and left overnight.

Living cell populations was determined using Cell Titer 96non-Radioactive Cell Proliferation Assay Kits (Promega, Madison, Wis.).The kits measure the amount of Formazan produced by metabolic conversionof Owen's reagent,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium,inner salt (MTS), by a dehydrogenase present in the mitochondria ofmetabolically active cells and is directly proportional to the number ofliving cells. Briefly, after the incubations with a test compound atvarious concentrations for 2 days, the culture medium was replaced withMTS/phenazine methosulfate in DMEM. After a 2-hour incubation at 37° C.,the absorbance was measured with a plate reader at 490 nm. Data wereexpressed as the percentage of control cells cultured in the absence ofany test compounds (as 100%).

Cytotoxicity of compounds 1-15 were tested. Unexpectedly, none of themexhibited inhibitory effect on cell growth at a concentration equal tothe EC₅₀ concentration against severe acute respiratory syndrome virus.Further, 10 of the test compounds exhibited no inhibitory effect on cellgrowth at a concentration four times as high as the EC₅₀ concentrationagainst severe acute respiratory syndrome virus.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating a subject suffering from aviral infection caused by a severe acute respiratory syndrome virus,comprising administering to the subject an effective amount of acomposition consisting of a pharmaceutically acceptable carrier and acompound of formula (I):

wherein R₁ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, or OR_(a); each of R₂, R₃, R₄ andR₁₀, independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, or aryl; each of R₅ and R₁₁,independently, is alkyl substituted with aryl; each of R₆, R₇, R₈, andR₉, independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, or OR_(b); and R₁₂ is C₁-C₁₀ alkylsubstituted with OR_(c), NHC(O)R_(c), or NHC(O)OR_(c); in which each ofR_(a), R_(b), and R_(c), independently, is H, C₁-C₁₀ alkyl, C₃-C₂₀cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl, or aryl; or a saltthereof.
 2. The method of claim 1, wherein R₁ is C₁-C₁₀ alkyl, C₃-C₂₀heterocycloalkyl, heteroaryl, aryl, or OR_(a); each of R₂, R₃, R₄ andR₁₀, independently, is H or C₁-C₁₀ alkyl; and each of R₆, R₇, R₈, andR₉, independently, is H or OR_(b).
 3. The method of claim 2, whereineach of R₅ and R₁₁ is alkyl substituted with phenyl.
 4. The method ofclaim 3, wherein R₂ is isopropyl.
 5. The method of claim 4, wherein eachof R₆, R₇, R₈, and R₉, independently, is H or OH.
 6. The method of claim5, wherein R₁ is C₃-C₂₀ heterocycloalkyl, heteroaryl, aryl, OR_(a), orC₁-C₁₀ alkyl substituted with a substituent selected from the groupconsisting of C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, heteroaryl,aryl, OR, SR, and NHC(O)OR; R being H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl,C₃-C₂₀ heterocycloalkyl, heteroaryl, or aryl.
 7. The method of claim 6,wherein the compound is


8. The method of claim 7, wherein the compound is


9. The method of claim 1, wherein the subject further suffers from aninfection caused by a human immunodeficiency virus.
 10. The method ofclaim 9, wherein the compound is


11. The method of claim 1, wherein the subject further suffers from aninfection caused by a feline immunodeficiency virus.
 12. The method ofclaim 11, wherein the compound is